Micro-chamber is a container in which minute reactions of up to several micro liters occur. This micro-chamber can be composed of silicon wafer, glass, metal or plastic. Micro-chamber plate is a plate in which the said micro-chambers are arranged 2-dimensionally. In this plate, inlets for sample input are located on one side and the other side is made of a transparent material for observation of inside reaction.
To measure the amount of a gene, real-time PCR has been developed to be co-executed with polymerase chain reaction (PCR) that facilitates the measurement of fluorescence increasing in proportion to the amount of a gene.
In real-time PCR, fluorescence generated from the PCR product is measured in every cycle and a certain cycle that gives at least required fluorescence is confirmed, leading to quantification of an early concentration of a specific gene.
Real-time PCR does not require electrophoresis upon completion of PCR and is rather performed along with PCR and facilitates quantification of the product, precisely it enables quantification of a gene having a specific nucleotide sequence in the concentration range of at least 109 (“A-Z of Quantitative PCR” edited by Stephen A. Bustin 2004-2006 International University, “Realtime PCR” edited by M. Tevfik Dorak 2006 Taylor & Francis Group).
Diverse devices for real-time PCR for analysis of multiple samples have been developed, which are exemplified by the apparatus analyzing 96 or 384 genes using a standard 96-well plate or 384-well plate (Roche Light cycler 480, ABI 7500, 7900).
The apparatus for real-time PCR provided by Roche requires the sample amount of 10-50 μl, which is rather a large amount.
To solve the above problem, different methods have been proposed to analyze many samples in a short period of time by reducing the amount of a sample by taking advantage of MEMS (Micro Electro Mechanical Systems) technique. One example is the method using a micro-chamber array plate.
The method using a micro-chamber array plate is composed of the following steps; loading a reaction sample in the micro-chamber; sealing each micro-chamber to isolate each reaction solution; inducing reaction and analyzing thereof.
Particularly, sample solution is added to the micro-chamber. The transparent micro-chamber plate for cell culture is covered with a semitransparent membrane to isolate micro-chambers one another. Only a cell is cultured in each micro-chamber. Then, culture medium is eliminated, to which Taqman reaction solution is added and sealed with clear oil to prevent evaporation. Fluorescence on the bottom of the plate is measured with temperature cycling (YASUDA, Kenji EP 1,541,678 A1, JP 2002245900 NUCLEIC ACID ANALYSIS CHIP AND NUCLEIC ACID ANALYZER).
According to the above method, different solutions are loaded in each micro-chamber by using a micropipette, which takes a long time. In particular, to inject samples into at least 1,536 micro-chambers, micro-automatic dispenser is necessary. This micro-automatic dispenser has to be washed after injection of each solution, which takes a long time. So, in fact, it is very difficult to use more than 384 plates.
Second, to overcome the above problem, Hdenori Nagai, a member of E. Tamiya group, proposed a reactor in which a micro-chamber array is composed of a silicon wafer by photolithography and chemical etching (Anal. Chem. 200173, 1043-1047, Development of a Microchamber Array for Picoliter PCR).
The said reactor uses microscope slide cover glass to prevent evaporation of PCR solution. But, when the cover glass is separated, there is a chance of cross contamination of the reaction solution. So, a water-repellent membrane is inserted in between the cover glass and a wafer. Then, the cover glass is eliminated first, and the water-repellent membrane is eliminated after drying the reaction solution, followed by analyzing. The above processes are troublesome. So, usability of this reactor for real-time gene quantitative amplification is limited.
Third, to overcome the problem according to the above quantitative amplification, Y. Matsubara et al of the same lab developed a micro-chamber array which was prepared by the steps of loading each primer in concave micro-chambers on a wafer using a microarray device; and drying thereof (7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems Oct. 5-9, 2003, Squaw Valley Calif. USA).
In this micro-chamber array, the upper part of the chip is covered with mineral oil to seal the micro-chamber completely, and then PCR reaction mixture is loaded on the mineral oil using a nano-jet dispenser. According to this method, 1,248 micro-chamber array chips having the volume of nano liter (0.65×0.65×0.2 mm) are prepared with a silicon wafer (1×3 inch) by photolithography and chemical etching. Primers and Taqman probe solution are distributed in the micro-chamber using a nano-liter dispenser, followed by drying. The whole chip is coated with mineral oil and thus each micro-chamber is separated and sealed.
The micro-chamber array prepared according to the third method above is advantageous for PCR without cross-contamination among reaction components. Particularly, according to this method, a mixed solution of Taq DNA polymerase and sample DNA is sprayed on top of the mineral oil, which is distributed in each micro-chamber, leading to PCR of each reaction component in the micro-chamber without cross-contamination.
However, the above method also has problems as follows. The method requires a microarray nano-liter dispenser for injection of a solution. Distribution takes a long time. And there is high possibility of cross-contamination among reaction solutions owing to the flow of mineral oil during moving the plate. In addition, bubbles are formed at high temperature during temperature cycling. And lens effect resulted from hydrophobicity of oil and aqueous solution that changes aqueous solution into a spherical shape is another problem which causes scattering and dispersion of excitation light and emitting light during optical measurement, making measurement error bigger.
Forth, PicoTiterPlate facilitating much more reactions than the third example above although the micro-chamber was also prepared by chemical etching like the third example was developed (John H. Leamon et al., A massively parallel PicoTiterPlate based platform for discrete pico-liter-scale polymerase chain reactions. Electrophoresis 2003, 24, 3769-3777).
According to this method, 300,000 independent PCR reactions occur at the same time with the amount of 39.5 pI.
But, this method requires a carrier for the fixation of primers/probes, which makes it inappropriate for real-time quantitative PCR asking even optical characteristics.
Fifth, ‘film reactor (or DNA card)’ was proposed (U.S. Pat. No. 5,948,673) to react micro-samples.
The film reactor is composed of three layers of thin films. Precisely, the lower layer film forms the bottom of the reactor, the middle layer film forms the side of the reactor and the upper layer film forms the sample inlets. After injecting micro sample solution using a pipette, the inlets have to be completely sealed for reaction. If the inlets are not completely sealed, the reaction mixture might be evaporated during PCR. To treat thousands of samples simultaneously, the said film reactor necessarily becomes so complicated. So, this reactor is hardly applied in reality.
Sixth, a reaction plate having a standard ELISA plate size and facilitating 1,536 fluorescence analysis reactions, was described in WO 02/40158 and U.S. Pat. No. 6,232,114.
As for the plate, multiple holes are penetrated in the plate and a transparent film with weak fluorescence is attached on the plate, forming multiple reaction vessels.
Reagent is loaded in the vessels, and the vessels are sealed with a transparent film, followed by reaction. This reaction plate has clear upper and lower parts. Excitation light is irradiated on one side and fluorescence is measured on the other side.
However, this sixth example also has problems. To analyze numbers of genes, different primers and probes have to be loaded in each micro-chamber. That is, thousands of different solutions have to be injected into such minute micro-chambers for analysis of numbers of samples. So, a special equipment like a nano-liter dispenser is required and the task takes a long time. In addition, high chance of malfunction of sample injection is another problem. Since the micro-chamber cannot be completely filled with the reaction mixture, water vapor is generated on the top of the micro-chamber during temperature rise, resulting in the difficulty in optical measurement.
Therefore, a novel micro-chamber plate is required which facilitates injection of samples in multiple micro-chambers evenly, excludes cross-contamination among reaction solutions, prevents bubble formation and water vapor condensation on the side of optical measurement, and thus facilitates real-time analysis of lights generated from the reaction products.